Iron overload syndromes remain important causes of heart and liver failure in patients with thalassemia or hereditary hemochromatosis, which are common genetic diseases worldwide. Current therapy for these conditions includes serial phlebotomy (hemochromatosis) or chelation therapy (thalassemia), both of which are associated with poor patient compliance because of inconvenience and significant side effects. Both thalassemia and hereditary hemochromatosis are associated with inappropriately low levels of hepcidin, the key regulator of intestinal iron absorption and cellular iron storage, resulting in excessive dietary iron absorption. Hepcidin, produced in response to tissue iron loading and inflammation, binds the iron exporter ferroportin1, causing internalization of both proteins and reduced iron release from enterocytes to the circulation and from macrophages to other tissues. We hypothesize that raising hepcidin expression will enhance the efficacy of chelation therapy in thalassemia by reducing dietary iron absorption and promoting storage of iron in the reticuloendothelial system and could eliminate the need for phlebotomy treatment in patients with hemochromatosis. We have previously demonstrated that ferroportin1 is required for iron export and iron cycling through enterocytes and macrophages in zebrafish. We have also demonstrated that the regulation of hepcidin expression in zebrafish embryos resembles that observed in mammalian systems, in that it is responsive to transferrin-bound iron and the BMP pathway. Recently, we have also discovered that treatment with the phytoestrogen, genistein, results in increased hepcidin transcript levels in zebrafish embryos and in human hepatocytes. We propose to screen for additional small molecules that regulate hepcidin expression by conducting a chemical screen in zebrafish embryos. To elucidate the mechanism of action of molecules identified in the screen, we will characterize the modulators' effects on mammalian cellular iron transport, chromatin immunoprecipitation, RNA expression, and signaling pathways. The best candidate regulators will be evaluated in mouse models of thalassemia and hemochromatosis for their effects on iron overload and erythropoiesis. These studies will lead to the identification of molecules and pathways that may be adapted to generate treatments for patients with thalassemia or hereditary hemochromatosis.